Impact of flow rate on peak resolution in Size-Exclusion-Chromatography (SEC)

Temperature of the column can be used to improve resolution of the analytes due to better diffusion into the pores. Since separation on SEC columns is typically performed using non-denaturing or native conditions, column temperature seldom exceeds 40°C and most applications are run at ambient temperature. Improvement in resolution is usually achieved by varying the flow rates through the column rather than column temperature. Due to the separation principle described earlier, flow rate has to allow for diffusion of analytes into the pores. Generally, the lower the flow rate, the better the resolution, at least for large molecules. This is demonstrated in Figure 1, where flow rate was reduced gradually from 750 µL/min to 200 µL/min. Resolution between the peaks of oligonucleotides of 36, 18 and 9 nucleotide lengths increased correspondingly.

Figure 1: Effect of different flow rates on SEC separation

Table 1: Peak resolutions between peaks of 36mer and 18mer oligonucleotides (Rs1) and peaks of 18mer and 9mer oligonucleotides (Rs2) in SEC analyses using flow rates between 750 µL/min and 200 µL/min.